The present invention relates to novel DNA sequences that encode for a branched-chain alpha-ketoacid dehydrogenase (BCKDH) complex of an organism belong to the genus Streptomyces. It also relates to the production of a Streptomyces avermitilis branched-chain alpha-ketoacid dehydrogenase (bkd)-deficient mutant by genetic engineering technology. The bkd-deficient mutant lacks branched-chain alpha-ketoacid dehydrogenase activity, and is useful for the fermentative production of novel (non-natural) avermectins.
S. avermitilis naturally produces eight distinct but closely related antiparasitic polyketide compounds named avermectins. The avermectin complex produced by S. avermitilis has four major components, A1a, A2a, B1a, and B2a, and four minor components, A1b, A2b, B1b, and B2b. The structure of the various components are depicted below. 
The avermectin polyketide structure is derived from seven acetate, five propionate molecules, and one alpha-branched-chain fatty acid molecule, which is either S(+)-2-methylbutyric acid or isobutyric acid. The designations xe2x80x9cAxe2x80x9d and xe2x80x9cBxe2x80x9d refer to avermectins wherein the 5-substituent is methoxy or hydroxy, respectively. The numeral xe2x80x9c1xe2x80x9d refers to avermectins wherein a double bond is present at the 22-23 position, and numeral xe2x80x9c2xe2x80x9d to avermectins having a hydrogen at the 22-position and hydroxy at the 23-position. Lastly, the C-25 has two possible substituents: the sec-butyl substituent (derived from the incorporation of S(+)-2-methylbutyric acid) is present in the avermectin xe2x80x9caxe2x80x9d series, and the isopropyl substituent (derived from the incorporation of isobutyric acid) is present in the avermectin xe2x80x9cbxe2x80x9d series (for a review see Fisher, M. H. and Mrozik, H., 1984, xe2x80x9cMacrolide Antibioticsxe2x80x9d, Academic Press, chapter 14).
By xe2x80x9cnaturalxe2x80x9d avermectins is meant those avermectins produced by S. avermitilis wherein the 25-position substituent is, as mentioned above, either isopropyl or sec-butyl. Avermectins wherein the 25-position group is other than isopropyl or sec-butyl are referred to herein as novel or non-natural avermectins.
One metabolic route to the natural alpha-branched-chain fatty acids in their CoA form is from the alpha branched-chain amino acids isoleucine and valine through a branched-chain amino acid transaminase reaction followed by a branched-chain alpha-ketoacid dehydrogenase reaction. (Alternatively, branched-chain fatty acyl-CoA derivatives can arise from branched-chain alpha-ketoacids produced by de novo synthesis). These metabolic pathways are depicted below. 
A mutant of S. avermitilis with no detectable branched-chain alpha-ketoacid dehydrogenase (BCKDH) activity in the last mentioned enzyme was previously isolated (Hafner et al., 1988, European Patent EP 284,176, which issued on Oct. 20, 1993). The mutant was isolated following standard chemical mutagenesis of S. avermitilis strain ATCC 31272 in a screen searching for the absence of 14CO2 production from 14C-1 labeled 2-oxoisocaproic acid substrate (leucine analog). The mutant is unable to synthesize natural avermectins except when the S(+)-2-methylbutyric acid or isobutyric acid or a precursor bearing the isopropyl or sec-butyl (S-form) group is added to the medium in which the mutants are fermented. The mutant is also capable of producing novel (non-natural) avermectins when fermented under aqueous aerobic conditions in a nutrient medium containing an exogenously added alternative carboxylic acid, such as cyclohexane carboxylic acid (CHC), or a precursor thereof, as indicated above.
To clone the genes that encode the branched-chain alpha-ketoacid dehydrogenase complex of S. avermitilis is highly desirable. Manipulation of these genes through recombinant DNA techniques should facilitate the production of natural and novel avermectins. For certain strains, increased titer of natural avermectins would be anticipated by increasing the copy number of the bkd genes. In addition, generation of an irreversibly blocked bkd strain, having BCKDH activity permanently deleted or modified by gene replacement, would be an improved alternative to the bkd mutant which was obtained, as mentioned before, by chemical mutagenesis.
The alpha-ketoacici dehydrogenase multienzyme complexesxe2x80x94the branchedxe2x80x94chain alpha-ketoacid dehydrogenase (BCKDH) complex, the pyruvate dehydrogenase (PDH) complex, and the alpha-ketoglutarate dehydrogenase (KGDH) complex catalyze the oxidative decarboxylations of branched-chain alpha-ketoacids, pyruvate, and alpha-ketoglutarate, respectively, releasing CO2 and generating the corresponding Acyl-CoA and NADH (Perham, R. N., 1991, Biochemistry, 30: 8501-8512). Each complex consists of three different catalytic enzymes: decarboxylase (E1), dihydrolipoamide acyltransferase transacylase (E2), and dihydrolipoamide dehydrogenase (E3).
Branched-chain alpha-ketoacid dehydrogenase (BCKDH) is a multienzyme complex composed of three functional components, E1, the decarboxylase, E2, the transacylase, and E3, the lipoamide dehydrogenase. The purified complexes from Pseudomonas putida, Pseudomonas aeruginosa, and Bacillus subtilis, are composed of four polypeptides. The purified mammalian complexes also consist of four polypeptides, E1alpha, E1beta, E2, and E3. An alpha-ketoacid dehydrogenase complex has been isolated from Bacillus subtilis which has both pyruvate and branched-chain alpha-ketoacid dehydrogenase activities. This dual function complex oxidizes both pyruvate branched-chain alpha-ketoacids for membrane phospholipids.
Cloning of prokaryotic branched-chain alpha-ketoacid dehydrogenase genes has been reported for Pseudomonas and Bacillus. In these systems it was found that the genes encoding the BCKDH were clustered in an operon. The genes of the BCKDH complex of Pseudomonas putida have been cloned and the nucleotide sequence of this region determined (Sykes et al., 1987, J. Bacteriol., 169:1619-1625, and Burns et al., 1988, Eur. J. Biochem, 176:165-169, and 176:311-317). The molecular weight of E1alpha is 45289, of E1beta is 37138, of E2 is 45134, and of E3 is 48164. The four genes are clustered in the sequence: E1alpha , E1beta, E2, and E3. Northern blot analysis indicated that expression of these four genes occurs from a single mRNA and that these genes constitute an operon. There is a typical prokaryotic consensus promoter immediately preceding the start of the E1alpha coding region that permits the constitutive expression of the Pseudomonas bkd genes. The initiator codon for the E1beta coding region is located only 40 nucleotides downstream from the end of the E1alpha open reading frame (ORF). In contrast, there is no intergenic space between the E1beta and E2 ORFs since the stop codon for the E1beta ORF is the triplet immediate preceding the initiator codon of the E2 ORF. The intergenic space between the E2 and the E3 ORFs is reduced to only 2 nucleotides. Therefore, the Pseudomonas bkd genes are tightly linked.
Similarly, the operon coding for the Bacillus subtilis BCKDH/PDH dual complex has been cloned (Hemila et al., 1990, J. Bacteriol., 172:5052-5063). This operon contains four ORFs encoding four proteins of 42, 36, 48, and 50 kilodaltons (kDa) in size, shown to be highly homologous to the E1alpha, E1beta, E2, and E3 subunits of the Pseudomonas bkd cluster. The genes encoding the alpha and beta subunits of the E1 component of the dual BCKDH/PDH multienzyme complex from Bacillus stearothermophilus have also been cloned and sequenced (estimated molecular weights of the alpha and beta subunits are approximately 41,000 and 35,000, respectively) (Hawkins et al., 1990, Eur. J. Biochem., 191:337-346).
Additionally, the sequences of a number of eukaryotic E1 alpha and beta BCKDH subunits (human, bovine, and rat) have been disclosed. Recently, an amino acid sequence comparison of all the published sequences known for both E1alpha and E1beta components of the PDH and the BCKDH complexes from multiple species was performed by computer analysis (Wexler et al., 1991, FEBS Letters, 282:209-213). Interestingly, several regions of the alpha and beta subunits were identified that are highly conserved not only in all PDHs so far described, but also in both prokaryotic and eukaryotic BCKDH complexes.
Also, recently, three genes encoding, respectively, the alpha (bkdA) and beta (bkdB) subunits of the E1 component, and the E2 (bkdC) component of a BCKDH complex from Streptomyces avermitilis were cloned, sequenced, and analyzed by using a heterologous gene expression system (Denoya, C. D., 1993, xe2x80x9cCloned genes encoding branched-chain alpha-ketoacid dehydrogenase complex from Streptomyces avermitilisxe2x80x9d, U.S. patent application Ser. No. 08/100,518, filed Jul. 30, 1993). DNA sequence analysis showed the presence of putative transcriptional promoter sequences and bkd structural genes arranged as a cluster organized as follows: promoter sequence, E1-alpha (bkdA), E1-beta (bkdB), and E2 (bkdB) open reading frames. Additionally, the complete S. avermitilis bkdABC gene cluster was cloned downstream of the strong Escherichia coli T7 promoter for expression in an E. coli host. Similarly, the E1-alpha and E1-beta open reading frames (ORFs) were also cloned, either separately or together, downstream of the T7 promoter and each construction was tested for expression. These studies demonstrated that at least 2 open reading frames of the S. avermitilis bkd gene cluster (E1-alpha [bkdA] and E1-beta [bkdB]) were fully translatable when expressed in E. coli. In addition, enzymatic assays aimed to analyze specifically the E1 component of the BCKDH complex confirmed conclusively that two of the recombinant E. coli clones, one carrying the whole bkd gene cluster and other carrying together the E1-alpha and the E1-beta ORFs, contained E1 BCKDH-specific enzyme activity.
The present invention relates to the molecular cloning and analysis of a second cluster of novel genes encoding branched-chain alpha-ketoacid dehydrogenase (BCKDH) of Streptomyces avermitilis. The cluster contains at least 3 genes (bkdF, bkdG, and bkdH) encoding, respectively, for the E1-alpha, E1-beta and E2 subunits of the S. avermitilis BCKDH complex. The bkd gene cluster disclosed here is located approximately 12 kilobases (kb) downstream of the first bkd cluster (bkdA, bkdB, and bkdC genes) recently reported (Denoya, C. D., 1993, U.S. patent application Ser. No. 30 08/100,518, filed Jul. 30, 1993). Both clusters share a similar gene organization, and they are oriented in the same direction on the S. avermitilis chromosome. The corresponding structural genes in both clusters, though highly homologous, are different.
In addition, this invention also relates to the construction of a bkd mutant by genetic engineering technology. The bkd mutant, which carries a chromosomal deletion affecting the bkdF gene, lacks branched-chain alpha-ketoacid dehydrogenase activity, is unable to grow on isoleucine, leucine, and valine as sole carbon sources, and is also incapable of making natural avermectins in a medium lacking both S-2-methylbutyric and isobutyric acids. The mutant disclosed here is useful to produce novel (non-natural) avermectins through fermentation in a medium containing an appropriate alternative carboxylic acid, such as cyclohexane carboxylic acid (CHC). Further, this invention relates to the construction of a mutant that carries a chromosomal deletion affecting both the bkdF gene and the bkdABC gene cluster.
Streptomyces has one major genetic linkage group, one chromosome, that is frequently present in multiple copies per hyphal compartment but is present only as a single copy in the spores. The Streptomyces genome is characteristically large among prokaryotes (5xc3x97103 to 7xc3x97103 kilobases [kb]) (Gladek, A., and Zakrzewska, J., 1984, FEMS Microbiol. Lett., 24:73-76), about two times the size of that of E. coli. 
One particularly interesting aspect of the Streptomyces genetics is the frequent chromosomal rearrangements involving extensive deletions which are frequently accompanied by intense DNA amplifications (Birch A. et al, 1990, J. Bacteriol., 172:4138-4142). Amplifications and deletion events in Streptomyces are two to three orders of magnitude larger than similar events in E. coli and B. subtilis. Deletions constituting 18% of the chromosome and amplifications representing 45% of the genome have been reported. Despite their inherent long term instability, duplications of certain genes arise with frequencies as high as one in 104 cells. Such duplications are usually generated by illegitimate crossing-over events that involve short, partially homologous sections of DNA in dividing daughter chromosomes. Such duplication events, by creating new stretches of DNA on the same chromosome, automatically tend to be followed by further gene amplification or elimination events. Multiple copies not only of the same gene but also of two different genes with highly similar structures will tend to recombine and to produce deletions. A gene""s inherent stability thus demands that it not be too similar to any other genes.
The presence of multiple copies of a gene or group of genes in Streptomyces is not unusual. A modular organization of genes required for synthesis of the polyketide portion of the macrolide antibiotic erythromycin in Saccharopolyspora erythraea has been reported (Donadio et al., 1991, Science, 252:675-679).
We have discovered that Streptomyces avermitilis has at least 2 clusters of bkd genes: one cluster comprising bkdA, bkdB, and bkdC genes, the other cluster comprising bkdF, bkdG, and bkdH genes. We speculate that both the bkdABC (disclosed previously in U.S. patent application Ser. No. 08/100,518, filed Jul. 30, 1993 and referred to above) and the bkdFGH gene clusters disclosed here arose by gene duplication in S. avermitilis, and that the 2 copies accumulated, through mutations, enough differences to assure survival (avoiding recombination/deletion events).
In addition to disclosing a second novel cluster of genes encoding BCKDH, we also disclose the construction of a Streptomyces avermitilis bkdF mutant by genetic engineering technology. The bkdF mutant lacks branched-chain alpha-ketoacid dehydrogenase activity, is unable to grow with isoleucine, leucine, and valine as sole carbon sources, and is also incapable of making natural avermectins in a medium lacking both S-2-methylbutyric and isobutyric acid. The bkdF mutant is useful for producing novel avermectins through fermentation in a medium containing an appropriate alternative carboxylic acid, such as cyclohexane carboxylic acid (CHC). We also disclose the construction of a mutant that carries a chromosomal deletion affecting both the bkdF gene and the bkdABC gene cluster.
Generation of Streptomycetes mutants using chemical and physical mutagens is an important technology to analyze gene function and regulation, and to develop improved industrial strains. Recent developments in Streptomyces gene cloning have resulted in the possibility of manipulating by recombinant DNA technology a myriad of cloned genes such as resistance, biosynthetic, and regulatory genes, and to elucidate gene organization and regulation. Another mutagenic approach, gene replacement, was successfully applied in Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis (Scherer, S. and R. W. Davis, 1979, Proc. Natl. Acad. Sci. U.S.A., 76: 4951-4955; Shortle, D., et al., 1982, Science, 217:371-373; Stahl, M. L., and Ferrari, E., 1984, J. Bacteriol., 158:411-418), and also in Saccharopolyspora erythraea (Weber, J. M., and Losick, R., 1988, Gene, 68:173-180) and several streptomycetes species. This technique allows the introduction of an in vitro generated mutation carried on a plasmid into the chromosome of the host strain. This approach is based on the findings that recombination occurs between the host chromosome and a plasmid containing a homologous region.
Gene replacement in S. avermitilis occurs by means of homologous recombination between cloned sequences carried in the vector and their chromosomal counterparts. Presumably, two crossovers occurring simultaneously, or a single crossover leading to integration and a subsequent resolution step where the integrated plasmid is excised, cause reciprocal exchange between the cloned and resident sequences. We have observed both double and single crossover events in S. avermitilis. Both mechanisms, double crossover and single crossover, followed by excision result in the same product. By using this approach we were able to disrupt the E1-alpha open reading frame of the bkdF gene of S. avermitilis. The disruption involved a chromosomal deletion of about 1.4 kb affecting the 5xe2x80x2-half of the gene encoding the E1-alpha subunit of the BCKDH complex. The resulting mutant strain, which exhibits all the characteristic phenotypical traits of a bkd mutant, is stable and can be used to generate valuable novel avermectins by fermentation.
All publications cited in this document are incorporated herein by reference in their entireties.
Technical terms used throughout this application are well known to those skilled in the art of molecular genetics. Terms frequently utilized in this invention are defined below:
Amplification: Refers to the production of additional copies of a chromosomal sequence, found as either intrachromosomal or extrachromosomal DNA.
Antibiotic Resistance Gene: DNA sequence that conveys resistance to an antibiotic when introduced into a host cell that is naturally sensitive to that particular antibiotic. Also known as antibiotic marker.
Clone: Large number of cells or molecules identical with a single ancestor.
Cloning Vector: Any plasmid into which a foreign DNA may be inserted to be cloned. It carries foreign DNA into a host bacterial cell upon transformation.
CoA: Coenzyme A.
Cohesive End Sequence (Cos): DNA sequence derived from bacteriophage lambda allowing in vitro packaging.
Cosmid: Plasmid into which bacteriophage lambda cos sites have been inserted; as a result, the plasmid DNA (carrying foreign DNA inserts) can be packaged in vitro in the phage coat.
cRNA: Single-stranded RNA complementary to DNA, synthesized from the latter by in vitro transcription.
Crossover: Refers to the point where a reciprocal exchange of material between a cloned sequence and its chromosomal counterpart occurs.
Dalton: unit of mass commonly used in connection with molecular dimensions corresponding to one hydrogen atom.
DNA Ligation: The formation of a chemical bond linking two fragments of DNA.
Double Crossover Events: Two crossovers occurring either simultaneously or in succession. As a result, a reciprocal exchange between the cloned and resident sequences occurs.
Gene Cluster: A group of genes physically close on the chromosome.
Gene Replacement: Technique which permits the introduction of an in vitro derived mutation carried on a plasmid into the chromosome. The replacement occurs when the host chromosome and a plasmid containing a region homologous to it recombine.
Genome: Entire chromosome set. The sum total of all of an individual""s genes.
Hybridization, Colony Hybridization: Technique used to identify bacterial colonies carrying chimeric vectors whose inserted DNA is similar to some particular sequence.
Hypha: The principal element of the growing or vegetative form of a mold. Hyphae are tubular structures, about 2 to 10 microns in diameter, that form a mass of intertwining strands called mycelium.
kb: Abbreviation for 1,000 base pairs of DNA or RNA.
Linker: Short synthetic duplex oligodeoxynucleotide containing the target site for one or more restriction enzymes. It is added to a vector to create a novel polylinker or multiple cloning site (MCS).
NADH: Reduced nicotinamide adenine dinucleotide.
Nucleotide: building block, or monomeric unit, of nucleic acids.
Oligonucleotide: A short chain of nucleotides.
Operon: A complete unit of bacterial gene expression and regulation, including structural genes, regulator genes, and control elements in DNA recognized by regulator gene product(s).
Plasmid: Autonomous, self-replicating, extrachromosomal circular DNA.
Plasmid Copy Number: Number of plasmid molecules maintained in bacteria for every host chromosome.
Primer: Short sequence of DNA or RNA that is paired to one strand of DNA and provides a free 3xe2x80x2-hydroxy end at which a DNA polymerase starts synthesis of a deoxyribonucleotide chain.
Prokaryotic Cells: The small, relatively simple cells comprising most microorganisms.
Promoter: Region of DNA responsible for the initiation of transcription.
Restriction Enzyme: Enzyme that recognizes a specific short sequence of DNA and cleaves it.
Restriction Recognition Sequence: DNA sequence specifically recognized by a particular restriction enzyme. Also known as target site.
Shuttle Vector: Bifunctional cloning vector able to replicate in one or more alternative hosts (e.g., E. coli and Streptomyces).
Single Crossover Event: Single reciprocal genetic recombination occurring at a single point. It causes integration of an incoming circular plasmid or phage vector and a duplication in the homologous chromosome sequence.
Southern Blotting: The procedure for transferring denatured DNA from an agarose gel to a nitrocellulose filter where it can be hybridized with a complementary nucleic acid probe.
Subcloning: Transferring cloned fragments of DNA from one type of vector to another, for example, from a recombinant cosmid to a plasmid. The new recombinant plasmid is then transformed into an appropriate host cell to produce a subclone strain.
Transformation of Bacterial Cells: Describes the acquisition of new genetic markers by incorporation of added DNA.
This invention relates to an isolated DNA segment that encodes for a branched-chain alpha-ketoacid dehydrogenase complex of an organism belonging to the genus Streptomyces.
This invention also relates to an isolated DNA segment, as described above, that further comprises a DNA region that regulates the expression of such branched-chain alpha-ketoacid dehydrogenase complex.
This invention also relates to an isolated DNA segment that encodes for a Streptomyces avermitilis branched-chain alpha-ketoacid dehydrogenase complex.
This invention also relates to a DNA segment comprising the DNA sequence of SEQUENCE ID NO. 1, SEQUENCE ID NO. 2, SEQUENCE ID NO. 3 or SEQUENCE ID NO. 4, as described below, or an allelic variation of such sequence. It also relates to a DNA segment that is a subset of the foregoing DNA segment and functionally equivalent to it.
This invention also relates to: (a) recombinant DNA comprising the DNA sequence of SEQUENCE ID NO. 1, SEQUENCE ID NO. 2, SEQUENCE ID NO. 3 or SEQUENCE ID NO. 4, or an allelic variation of such sequence; (b) a plasmid comprising such recombinant DNA; and (c) a host cell into which such recombinant DNA has been incorporated.
This invention also relates to the genes for branched-chain alpha-ketoacid dehydrogenase complex contained in a DNA segment selected from the group consisting of pCD713, pCD740, pCD747 and pCD854, as defined below.
This invention also relates to a DNA segment comprising the DNA sequence of SEQUENCE ID NO. 1, SEQUENCE ID NO. 2, SEQUENCE ID NO. 3 or SEQUENCE ID NO. 4, or an allelic variation of such sequence.
This invention also relates to a DNA segment comprising a DNA sequence that is a subset of the DNA sequence of SEQUENCE ID NO. 1, SEQUENCE ID NO. 2, SEQUENCE ID NO. 3 or SEQUENCE ID NO. 4, or an allelic variation thereof, and that is capable of hybridizing to, respectively, SEQUENCE ID NO. 1, SEQUENCE ID NO. 2, SEQUENCE ID NO. 3 or SEQUENCE ID NO. 4, or an allelic variation thereof, when used as a probe, or of amplifying all or part of such sequence when used as a polymerase chain reaction primer.
This invention also relates to a substantially purified polypeptide comprising the amino acid sequence of SEQUENCE ID NO. 5, SEQUENCE ID NO. 6, SEQUENCE ID NO. 7 or SEQUENCE ID NO. 8.
This invention also relates to a method of producing a natural avermectin, comprising fermenting, under conditions and in a fermentation medium suitable for producing such natural avermectin, S. avermitilis in which the copy number of a genomic fragment comprising one or more of the bkdF, bkdG and bkdH genes has been increased.
This invention relates to a method of producing a natural avermectin, comprising fermenting, under conditions and in a fermentation medium suitable for producing such natural avermectin, S. avermitilis in which expression of a genomic fragment comprising one or more of the bkdF, bkdG and bkdH genes has been enhanced by manipulation or replacement of the genes responsible for regulating such expression.
This invention also relates to a method of producing a novel avermectin, comprising fermenting, under conditions and in a fermentation medium suitable for producing such novel avermectin, S. avermitilis in which expression of a genomic fragment comprising one or more of the bkdF, bkdG and bkdH genes has been decreased or eliminated by deletion, inactivation, replacement or other manipulation of the genes responsible for such expression.
A preferred embodiment of the invention relates to the foregoing method of producing a novel avermectin, which comprises fermenting, under conditions and in a fermentation medium suitable for producing such novel avermectin, S. avermitilis in which a genomic fragment comprising one or more of the bkdF, bkdG, and bkdH genes has been deleted or inactivated.
This invention also relates to a method of producing a novel avermectin, comprising fermenting, under conditions and in a fermentation medium suitable for producing such novel avermectin, S. avermitilis in which expression of a genomic fragment comprising one or more of the bkdA, bkdB and bkdC genes and one or more of the bkdF, bkdG and bkdH genes has been decreased or eliminated by deletion, inactivation, replacement or other manipulation of the genes responsible for such expression.
A preferred embodiment of the invention relates to the foregoing method of producing a novel avermectin, which comprises fermenting, under conditions and in an fermentation medium suitable for producing such novel avermectin, S. avermitilis in which a genomic fragment comprising one or more of the bkdA, bkdB and bkdC genes and one or more of the bkdF, bkdG and bkdH genes has been deleted or inactivated.